TY - JOUR
T1 - Use of 3-aminotyrosine to examine the pathway dependence of radical propagation in Escherichia coli ribonucleotide reductase
AU - Minnihan, Ellen C.
AU - Seyedsayamdost, Mohammad R.
AU - Stubbe, Joanne
PY - 2009/12/29
Y1 - 2009/12/29
N2 - Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 50-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y122 •) cofactor which, in the presence of S and E, oxidizes C 439 in α to a thiyl radical, C439•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y122• → W48 → Y356 in β2 to Y731 → Y730 → C439 in α2. 3-Aminotyrosine (NH2Y) has been sitespecifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH2Y•) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y 356F-β2:Y731NH2Y-α2, Y 356F-β2:Y730NH2Y-α2, and wt-β2:Y731F/Y730NH2Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH2Y• was detected. To study off-pathway oxidation, Y413, located 5Å from Y 730 and Y731 but not implicated in long-range oxidation, was examined. Evidence for NH2Y413• was sought in three complexes: wt-β2:Y413NH2Y-α2 (a), wt-β2:Y731F/Y413NH2Y-α2 (b), and Y356F-β2: Y413NH2Y-α2 (c). With (a), NH2Y• was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant forNH2Y• formation was 0.2-0.3% of that observed at 731 and 730, and with (c), noNH 2Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C439.
AB - Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 50-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y122 •) cofactor which, in the presence of S and E, oxidizes C 439 in α to a thiyl radical, C439•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y122• → W48 → Y356 in β2 to Y731 → Y730 → C439 in α2. 3-Aminotyrosine (NH2Y) has been sitespecifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH2Y•) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y 356F-β2:Y731NH2Y-α2, Y 356F-β2:Y730NH2Y-α2, and wt-β2:Y731F/Y730NH2Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH2Y• was detected. To study off-pathway oxidation, Y413, located 5Å from Y 730 and Y731 but not implicated in long-range oxidation, was examined. Evidence for NH2Y413• was sought in three complexes: wt-β2:Y413NH2Y-α2 (a), wt-β2:Y731F/Y413NH2Y-α2 (b), and Y356F-β2: Y413NH2Y-α2 (c). With (a), NH2Y• was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant forNH2Y• formation was 0.2-0.3% of that observed at 731 and 730, and with (c), noNH 2Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C439.
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U2 - 10.1021/bi901439w
DO - 10.1021/bi901439w
M3 - Article
C2 - 19916558
AN - SCOPUS:73149099389
SN - 0006-2960
VL - 48
SP - 12125
EP - 12132
JO - Biochemistry
JF - Biochemistry
IS - 51
ER -